1 Clostridium species Gram positive large rods Resistant spores; spores usually bulge the mother cell Strict anaerobes (prefer 2-10% CO 2 ) Most Spp are motile; C. Perfringens is not Biochemically active. However, oxidase – ve, and catalase -ve Produce volatile fatty acids Produce enzymes including proteases and saccharases Produce toxins – Tetanus toxin on plasmid, botulism toxin on lysogenic phage Antimicrobial sensitive Resistance may occur

2 Clostridia Present in soil, alimentary tract of animals and in faeces (normal flora) Many species, not all pathogens Pathogens grouped according to mode and sites of action of their potent toxins - neurotoxic clostridia, histotoxic clostridia, enteropathogenic and enterotoxaemia producing clostridia

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4 Gram stain of Clostridium : Gram-positive rods Ink Stain of Sporulating Clostridium : spores appear clear, vegetative cells dark

5 Neurotoxic Clostridia Tetanus – C. tetani Potentially fatal intoxication affecting many species including man. Species susceptibility varies – horses and man, highly susceptible, ruminants and pigs moderately so, carnivores comparatively resistant, poultry resistant.

6 Diagnosis Characteristically produces a terminal spherical endospore that bulges the cell giving the drumstick appearance Diagnosis is often based on case history and characteristic clinical signs For isolation, necrotic wound tissue can be used for inoculation on blood agar. Growth usually take 3-4 days at 37˚C under an atmosphere of H 2 and CO 2 C. tetani is haemolytic on blood agar (beta) and tend to have a spreading growth Diagnosis can be further confirmed by identifying the toxins

7 Clostridium tetani

8 Tetanus Tetanolysin (haemolysin) toxin does not seem to play a role in pathogensis. Neurotoxin tetanospasmin antigenically uniform (antibodies protect) Plasmid encoded toxin Infection occurs when endospores are introduced into tissue traumatised or not from soil or faeces. Vegetative bacteria develops from spore in wound Vegetative bacteria produce toxin Deep penetrating wounds in the horse Castration and docking in sheep Abrasions in cows and ewes Umbilicus in all animals

9 Tetanus - pathogenesis Tetanus toxin consists of 2 chains joined by disulphide bridge Light chain is toxic, heavy chain responsible for receptor binding and internalisation Binds irreversibly to ganglioside receptors on motor neuron terminals and transported to CNS by intra axonal flow Toxin transferred trans synaptically to its site of action in the terminals of inhibitory neurons where it blocks pre-synaptic transmission of inhibitory signals Hydrolyses synaptobrevins – proteins of vesicles containing neurotransmitters Inhibitory neurotransmitter (glycine and GABA) release is prevented results in SPASTIC paralysis Bound toxin not neutralised by antitoxin Mouse injected with Tetanus Toxin in Left Hind leg

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11 C.tetani, HORSE

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13 Neurotoxic Clostridia Botulism – C. botulinum Serious potentially fatal intoxication by pre-formed toxin Neurotoxins Germination of endospores, growth of vegetative cells and toxin production occurs in rotting carcasses, decaying vegetation and contaminated canned food Types C and D cause outbreaks in domestic animals Outbreaks in waterfowl, cattle, horses, sheep, mink, poultry, farmed fish

14 Botulism - pathogenesis Neurotoxins of C. botulinum are the most potent biological toxins known Botulinum toxin is synthesized as a single polypeptide chain; The toxin is then nicked by a bacterial protease to produce two chains, the nicked toxin A becomes, on a molecular weight basis, the most potent toxin found in nature. Preformed toxin in food is absorbed from GI tract and circulates in blood Acts at neuromuscular junctions of cholinergic nerves and peripheral autonomic synapses. Remains at junction Irreversible interference with acetylcholine resulting in FLACCID paralysis Death from paralysis of respiratory muscles

15 Botulism - Diagnosis Deep litter containing poultry carcasses on pasture involved Clinical signs and toxin detection are the key means of DX Detection of toxin in serum difficult – not much present, test GI contents in mouse assay Toxin types C and D (B has also affected cattle Risk to human food chain – meat, milk?

16 motor end plate Ach-containing vesicles C. botulinum toxin blocks release of Ach (cleave synaptobrevin or SNAP25) stimulation blocked Muscle fibre

17  Toxins that produce both tetanus and botulism are very similar in structure and function despite the almost diametrically opposed clinical manifestations of the diseases. This is because the toxins act at different sites  Both toxins bind neuromuscular junctions but TT transported via retrograde axonal transport to CENTRAL NERVOUS SYSTEM and brain stem. BT acts at PERIPHERAL cholinergic synapses  Both toxins substrates are 3 protein components of the docking complex by which synaptic vesicles fuse with the terminal cell membrane and release acetyl choline (BT) or inhibitory neurotransmitters (TT).  Proteins clamp the vesicle to the presynaptic membrane. Clostridial neurotoxins inhibit vesicle release by cleaving peptide bonds in these proteins. Each toxin has a specific locus of activity. Comparison of Tetanus and Botulism Neurotoxins

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19 Histotoxic Clostridia Variety of lesions in domestic animals Exotoxins produced by replicating bacteria induce local tissue necrosis and systemic effects which may be lethal Present in tissues as latent spores Introduced into wounds as mixed infection (gas gangrene)

20 Histotoxic Clostridia - pathogenesis Ingested endospores excreted but some may leave intestine and lie dormant in tissue Tissue injury leading to reduced oxygen tension required for germination and vegetative replication Local necrosis produced by exotoxins of replicating bacteria allows further proliferation and tissue damage Endogenous infections such as blackleg, infectious necrotic hepatitis and bacillary haemoglobinuria result from activation of dormant spores in muscle or liver Exogenous infections such as gas gangrene (myositis) result from introduction of clostridia into wounds. Anaerobic environment of necrotic tissue allows replication, local and systemic toxin production

21 Histotoxic Clostridia C. chauvoei Blackleg in cattle C. septicum Malignant oedema in cattle pigs and sheep, braxy (abomasitis) in sheep C. novyi type A Big head in rams, wound infection C. perfringens type A ---Necrotic enteritis in chickens, Gas gangrene (myositis) C. sordellii Myositis in cattle, sheep, horses, abomasitis in lambs C. novyi type B Black disease in sheep (necrotic hepatitis) C. haemolyticum Bacillary haemoglobinuria in cattle and occasionally sheep

22 Histotoxic Clostridia Cl. chauvoei Cl. novyi Type B Blackleg Black's Disease  commensal / ingestion  relocalisation to musclerelocalisation to liver  bruising fluke migration  spore germination and toxinogenesis  haemolysisoedema crepitationshocknecrosis

23 C. chauvoei -blackquarter case

24 C. novyi C.novyi – Liver lesions C. novyi in tissue

25 Histotoxic Clostridia Cl. septicum injury to abomasum wound  colonisation by inoculation with commensal/ingested clostridia Cl. septicum  toxinogenesis toxinogenesis (?)  localised mucosalcellulitis & myositis haemorrhage (malignant oedema) & necrosis   gangrene toxaemia Braxy

26 Histotoxic Clostridia - Control Treatment (antibiotics) usually ineffective unless given early Vaccination – bacterin + toxoid + adjuvant Multicomponent vaccines + boosters + annual re- vaccination

27 Enteropathogenic and enterotoxaemic Clostridia C. perfringens types A-E Replicate in intestinal tract and elaborate toxins which produce both localised and systemic effects Predisposing factors are inappropriate husbandry, dietary changes, local environmental influences

28 Enteropathogenic Clostridia C. perfringens types A-E produce a number of potent immunologically distinct exotoxins Cause local and systemic effects Pattern of toxin production varies with each C. perfringens type and determines clinical syndrome observed

29 Biotype Major toxins produced Disease association A  Gas gangrene of humans and animals, fowl and porcine necrotic enteritis, bovine and ovine enterotoxaemia, food poisoning in man, colitis in horses, canine haemorrhagic gastroenteritis B , ,  Lamb dysentery, enterotoxaemia of foals, sheep, goats C ,  Pig-bel (necrotic enteritis) in man, enterotoxaemia of sheep (struck), calves, lambs, piglets D ,  Enterotoxaemia of lambs and sheep (pulpy kidney), goats, cattle (human) E ,  Rabbit enteritis, enterotoxaemia of calves and lambs Association of C. perfringens biotypes with diseases of humans and animals

30 Enteropathogenic Clostridia C. perfringens TYPE A  toxin Enterotoxin Necrotic Enteritis in chickens Food poisoning TYPE B  toxin Lamb dysentery

31 TYPE C  toxin Piglets – haemorrhagic enteritis Enterotoxaemia in calves Struck in sheep TYPE D  toxin Pulpy kidney in fat lambs Enterotoxaemia in sheep TYPE E  toxin Enterotoxaemia in sheep C. perfringens

32 SPECIES OF CLOSTRIDIUM ASSOCIATED WITH DISEASE C. difficile Enteritis C. haemolyticum Bacillary Haemoglobinuria C. spiroforme Rabbit Diarrhoea C. colinum Quail disease

33 Clostridium difficile 25% of antibiotic associated diarrhoea in man (hospital acquired) Pseudomembranous colitis in man Diarrhoea in hamsters and guinea pigs Enteritis in neonatal pigs